CN110760494B - Preparation and application of a kind of monocyclic thorn autolysase - Google Patents
Preparation and application of a kind of monocyclic thorn autolysase Download PDFInfo
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Abstract
本发明属于生物制品技术领域，具体涉及一种单环刺螠自溶酶的制备及应用。取单环刺螠内脏用无菌水清洗并匀浆，加入PBS进行浸提，置于低温环境下静置过夜后离心收集上清；用饱和硫酸铵溶液在低温环境下梯度沉淀得到蛋白质沉淀，用PBS缓冲液重溶后，透析得到自溶酶粗酶溶液；粗酶溶液经柱层析分离纯化得单环刺螠自溶酶。通过本方法制备得到的单环刺螠自溶酶有良好的和热稳定性能，对小球藻等藻类细胞具有良好的破壁效果，可以运用到水产动物及加工副产物中活性物质的提取，以及包含藻类在内的食品生产加工中，从而提升降解效果，降低生产成本。
The invention belongs to the technical field of biological products, and in particular relates to the preparation and application of a monocyclic thorn autolyase. The viscera was washed with sterile water and homogenized, PBS was added for leaching, and the supernatant was collected by centrifugation after standing in a low temperature environment overnight; the protein precipitate was obtained by gradient precipitation with saturated ammonium sulfate solution in a low temperature environment, After redissolving with PBS buffer, the crude enzyme solution of autolysin was obtained by dialysis; the crude enzyme solution was separated and purified by column chromatography to obtain the autolysate of S. chinensis. The single-ringed thorn autolyase prepared by the method has good thermal stability and good wall-breaking effect on algal cells such as Chlorella, and can be used in the extraction of active substances from aquatic animals and processing by-products. And in the production and processing of food including algae, so as to improve the degradation effect and reduce the production cost.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to preparation and application of urechis unicinctus autolytic enzyme.
Background
Urechis unicinctus, commonly called as urechis unicinctus, is a tubular urechis animal living in intertidal zone with muddy bottom or coral reef zone at the seaside like other urechis organisms, and is mainly distributed in Weihai cities, smoke tables and the like at the coasts of yellow sea and Bohai sea. The urechis unicinctus is large in individuals, the meat taste is delicious, and the muscle of the body wall is rich in protein and various amino acids necessary for the human body. Since ancient times, the seafood food has higher economic value as a rare seafood food in China, Japan and Korean coastal areas. People traditionally eat only their body walls, discarding their internal organs. However, research finds that the waste urechis unicinctus viscera are rich in protein and fat, rich in Ca, Mg, Fe, Zn and other elements, and rich in EPA, DHA and DPA. The urechis unicinctus has higher nutritive value, but because the urechis unicinctus has high water content, the enzyme system is rich, autolysis hydrolysis can be carried out under certain conditions, and the phenomenon of dissolution is very obvious particularly in the death process of the urechis unicinctus, which is caused by the fact that the urechis unicinctus contains autolytic enzyme. The autolytic enzyme causes urechis unicinctus to lose a large amount of nutrient components in the processing process.
In recent years, with the progress of enzymology, researchers at home and abroad have conducted extensive and intensive studies on the autolysis of aquatic animals, particularly sea cucumbers, fishes, shrimps, shellfishes and the like and by-products of the processing thereof. Aquatic animals are easy to generate unique autolysis due to the characteristics of high water content and rich enzyme systems. The autolysis process of aquatic animals is complex, the biochemical change is mainly caused by the degradation of protein and structural tissues by endogenous enzyme systems, and the enzymes playing a role in the process comprise protease systems with multiple catalytic activities, lysosomal enzymes and the like. The preparation of bioactive peptides by autolytic enzymolysis technology has become a main technical means for preparing bioactive peptides of aquatic animals. In addition, the autolysis technology can be used for effectively recycling and utilizing the nutrient components in the aquatic product processing byproducts, so that the pollution to the environment is avoided while the value of the raw materials is maximally utilized.
In recent years, research on extraction and properties of autolytic enzymes such as sea cucumber and cod has been advanced at home and abroad, but preparation and property research of the autolytic enzymes in urechis unicinctus has not been reported for a while.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of urechis unicinctus autolytic enzyme.
In order to achieve the purpose, the invention has the technical scheme that:
a method for preparing urechis unicinctus autolytic enzyme comprises cleaning viscera of urechis unicinctus with sterile water, homogenizing, adding PBS, leaching, standing at low temperature overnight, centrifuging, and collecting supernatant; carrying out gradient precipitation by using a saturated ammonium sulfate solution in a low-temperature environment to obtain a protein precipitate, re-dissolving the protein precipitate by using a PBS (phosphate buffer solution), and dialyzing to obtain an autolytic enzyme crude enzyme solution; and separating and purifying the crude enzyme solution by column chromatography to obtain the urechis unicinctus autolytic enzyme.
Centrifuging the crude enzyme solution, collecting supernatant, purifying by column chromatography, eluting by eluent, measuring absorbance at 340nm by an enzyme-labeling instrument, and collecting autolytic enzyme solution with the molecular weight of 8-10 KD; wherein the eluent is PBS containing Zn ions of 0.010-0.015 mol/L. Washing the urechis unicinctus viscera with sterile water, homogenizing, adding a PBS (phosphate buffer solution) with the pH of 8.0-9.0 and the concentration of 0.1mol/L, leaching for 12-16h at the temperature of 4-8 ℃, standing overnight at the temperature of 4 ℃, centrifuging and collecting a supernatant; carrying out gradient precipitation at 4 ℃ by using a saturated ammonium sulfate solution to obtain a protein precipitate.
After standing overnight and centrifuging, adding ammonium sulfate into the supernatant to 20-30% of saturation, and placing the supernatant in an environment of 4-8 ℃ for 4-8 h; centrifuging at 12000r/min at 8000-; centrifuging at 8000-.
The chromatographic column adopted by the column chromatography is one of Sephadex G-100, Sephadex G-75 and Sephadex G-50.
The method specifically comprises the following steps:
1. preparing an urechis unicinctus autolytic enzyme crude enzyme solution:
(1) washing the inner organs of the urechis unicinctus with sterile water, homogenizing, adding PBS (phosphate buffer solution) for leaching, standing overnight in a low-temperature environment, centrifuging and collecting the supernatant;
(2) carrying out gradient precipitation by using a saturated ammonium sulfate solution in a low-temperature environment to obtain protein precipitate, re-dissolving by using a PBS buffer solution, and dialyzing to obtain an autolytic enzyme crude enzyme solution.
2. And (3) column chromatography separation and purification of autolytic enzyme:
(1) carrying out high-speed centrifugation on the crude enzyme solution of the autolytic enzyme obtained by dialysis to obtain a supernatant;
(2) purifying the obtained supernatant by column chromatography, eluting with eluent, measuring absorbance at 340nm with an enzyme-labeling instrument, and collecting to obtain autolytic enzyme solution;
(3) and (3) performing SDS-PAGE on the autolytic enzyme solution obtained in the step (2) to identify the purity and the size and identify the activity.
The eluent is formed by adding 0.010-0.015mol/L Zn ions into PBS with the concentration of 0.1mol/L, pH 8.0.0.
The urechis unicinctus autolytic enzyme prepared by the method has the molecular weight of 8-10KD, is high in purity and has good serine protease enzymatic activity.
An application of urechis unicinctus autolytic enzyme in preparation for aquatic animal processing or active substance extraction.
The invention has the beneficial effects that:
the invention prepares the autolytic enzyme of urechis unicinctus by the 2-step method, carries out the purification of the autolytic enzyme by the chromatography technology, has simple operation method and lower implementation cost. The urechis unicinctus autolytic enzyme prepared by the method has good enzymatic activity and thermal stability, has a good wall breaking effect on algae cells such as chlorella and the like, can be applied to the processing of aquatic animals, the extraction of active substances and the production and processing of algae medical food, thereby improving the medical effect and reducing the production cost.
Drawings
FIG. 1 is an SDS-PAGE analysis of urechis unicinctus autolytic enzyme according to the present invention.
FIG. 2 is a graph showing the measurement of the serine protease activity of urechis unicinctus autolytic enzyme mentioned in example 5 of the present invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the following examples which illustrate specific embodiments of the invention and are not therefore to be construed as limiting the invention to the scope of the examples.
Example 1: preparation of urechis unicinctus autolytic enzyme
1. Preparing an urechis unicinctus autolytic enzyme crude enzyme solution:
(1) thawing the urechis unicinctus waste viscera frozen and stored at-20 to-10 ℃ at normal temperature, and cleaning with sterile water to remove impurities after the urechis unicinctus waste viscera are completely thawed. Adding PBS (0.1 mol/l, pH8.0), homogenizing, standing at 4 deg.C overnight, centrifuging at 5000r/min for 8min, and collecting supernatant;
(2) adding ammonium sulfate into the collected supernatant to 20% saturation, and standing at 4 deg.C for 4 hr; centrifuging at 8000r/min for 10min, collecting supernatant, adding ammonium sulfate to the supernatant to 70% saturation, and standing at 4 deg.C for 4 hr; centrifuging at 8000r/min for 10min, collecting precipitate, adding 10 times volume of PBS buffer solution into the precipitate, and dialyzing with dialysis bag with cut-off molecular weight of 8KD-14KD to obtain crude enzyme solution of autolytic enzyme.
2. And (3) column chromatography separation and purification of autolytic enzyme:
(1) centrifuging the crude enzyme solution of the autolytic enzyme obtained after dialysis at 12000r/min for 10min to obtain a supernatant;
(2) separating and purifying the supernatant obtained in the step (1) by Sephadex G-100 column chromatography, eluting by PBS containing 0.015mol/L Zn ions, measuring absorbance at 340nm by an enzyme-labeling instrument and collecting to obtain an autolytic enzyme solution;
(3) the autolytic enzyme solution obtained in step (2) was identified by SDS-PAGE, showing that the size of autolytic enzyme was 8-10KD, as shown in FIG. 1.
Example 2: evaluation of dissolving activity of autolytic enzyme on urechis unicinctus body wall
Weighing dry urechis unicinctus body wall samples, respectively placing the dry urechis unicinctus body wall samples in 6 EP tubes with the volume of 1.5ml, and equally dividing the samples into an experimental group and a control group. The urechis unicinctus autolytic enzyme solution obtained in example 1 was diluted to 1mg/ml with PBS. 1ml of diluted autolytic enzyme solution is added into 3 tubes of an experimental group, PBS buffer solution with the same volume is added into a control group, reaction is carried out in a water bath at 50 ℃, the body wall is taken out every 2 hours, water is sucked, weighing is carried out, and finishing and analysis are carried out on weight data of the body wall. The consumption degree calculation formula is as follows: consumption = initial weight-weight after reaction/initial weight x 100%, the results are shown in table 1.
TABLE 1 comparison of the solubilizing effect of autolytic enzyme on the wall of urechis unicinctus
The results in Table 1 show that the average degradation rates of the groups without adding autolytic enzyme were 9.52%, 13.34% and 20.95% respectively at 2, 4 and 6h of the experiment, and the average degradation rates of the groups with 1mg of autolytic enzyme were 57.14%, 68.57% and 91.43%, respectively, which are improved by 6, 5.14 and 4.36 times compared with the control group. Compared with the control group, the addition of the urechis unicinctus autolytic enzyme prepared in the embodiment 1 has a good dissolving effect on the urechis unicinctus body wall.
Example 3 evaluation of thermostability of autolytic enzyme
The autolytic enzyme solution prepared in example 1 was diluted to 1mg/ml with PBS. 1ml of autolytic enzyme solution was added to each of 4 EP tubes, heated in a water bath at 24 deg.C, 80 deg.C, 90 deg.C and 100 deg.C for 1h, and 1ml of PBS was added to the blank set of EP tubes. Adding equal weight of dried urechis unicinctus wall into each tube, and reacting in water bath at 55 deg.C. Taking out the urechis unicinctus body wall after 1h, 2h and 3h of reaction respectively, sucking water, weighing, sorting the body wall weight data, and calculating the consumption rate according to the formula recorded in the embodiment 2, wherein the results are shown in table 2.
TABLE 2 evaluation results of thermostability of autolytic enzymes
As shown in the results of Table 2, the consumption rates of the body wall after treatment at 80, 90 and 100 ℃ for 3 hours are respectively reduced to 22.57 percent, 22.22 percent and 19.41 percent; but still has a better effect than the consumption rate of 9.87% in the blank group. It is demonstrated that the autolytic enzyme obtained in this example 1 can still maintain a good body wall dissolution activity and has a good thermal stability after being treated at a high temperature of 80-100 ℃.
Example 4 evaluation of the Effect of autolytic enzyme on algal cell disruption
Taking 1ml of the extract with the concentration of 1.2X 107Placing the chlorella solution of cells/ml in a 1.5ml EP tube, centrifuging at 8000r/min for 10min, taking out the precipitate, eluting with PBS buffer solution containing 0.015mol/L Zn ions, uniformly distributing into 6 1.5ml EP tubes, centrifuging at 12000r/min for 10min, and pouring off the supernatant. Taking 3 of the 1.5ml EP tubes, adding 1ml (concentration is 1 mg/ml) of the autolytic enzyme solution obtained in example 1, taking 3 tubes as a control group, and adding PBS buffer solution with the same volume; reacting for 1h in a water bath at 60 ℃, and taking out; and (4) sucking the liquid in the tube by using a pipette gun, dripping the liquid on a cell counting plate, observing and counting under a 40-time microscope, and sorting and analyzing the data. The calculation formula of the chlorella is as follows: the number of chlorella/ml is 80 small square cell total/80 × 400 × 10000 × dilution times.
The results are shown in table 3, the number of intact chlorella observed in the experimental group is significantly reduced compared to the number of intact chlorella in the control group, which indicates that the autolytic enzyme obtained in example 1 has a better effect of breaking the wall of the algae cells.
Example 5: serine protease Activity assay
The autolytic enzyme solution prepared in example 1 was diluted to 1mg/ml with PBS. 0.05g of fluorescent casein is weighed and dissolved in 10ml of phosphate buffer solution with pH7.4, 10 mul of autolytic enzyme solution is added into 500 mul of 0.5% fluorescent casein solution, and the solution is averagely divided into 8 parts, each of which is 60 mul. The temperature is maintained at 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C and 80 deg.C for 15 min. Adding 10% trichloroacetic acid with the same volume, standing at 4 deg.C for 15min, centrifuging, and collecting supernatant. Absorbance was measured at 350 nm. And calculating the relative activity of the autolytic enzyme at each temperature by taking the highest absorbance as 100 percent. As shown in fig. 2. As can be seen, the urechis unicinctus autolytic enzyme provided by the invention has better serine protease activity, and can still keep about 60% of activity at 80 ℃.
The urechis unicinctus autolytic enzyme prepared by the method has good thermal stability, has good wall breaking effect on chlorella algae cells, can be applied to processing of aquatic animals and byproducts, extraction of active substances, and production and processing of algae medical food, and reduces production cost.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
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| CN101565696B (en) * | 2009-06-05 | 2011-09-14 | 大连工业大学 | Method for separating and purifying intestinal tract cathepsin B of sea cucumber |
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